Long-read direct infrared sequencing of crude PCR products for prediction of resistance to HIV-1 reverse transcriptase and protease inhibitors

Abstract

Patients infected with human immunodeficiency virus type 1 (HIV-1) are being treated with a number of different combinations of antiretroviral compounds that target the essential viral enzymes reverse transcriptase and protease. Different sets of HIV-1 mutations that confer drug resistance have been well defined; they allow reasonable prediction of the drug sensitivity pattern from analysis of the HIV-1 genotype in vivo. Since periodical monitoring of genotypic resistance is expected to improve clinical management in a large number of infected patients, practical and cost-effective methods are highly desirable to set at least medium-scale sequencing in clinical diagnostic settings. We present a complete protocol for direct sequencing of HIV-1 reverse transcriptase and protease-coding regions. Features making the system amenable to routine clinical use include: 1. Highly robust presequencing steps (plasma RNA extraction, reverse transcription, and nested PCR); 2. Direct use of the crude unpurified PCR product as the sequencing template; and 3. Use of infrared-labeled sequencing primers consistently allowing long reads, thus obviating the need for sequencing of both DNA strands.

An algorithm‐based genotypic resistance score is associated with clinical outcome in HIV‐1‐infected adults on antiretroviral therapy

“…A nested PCR was used to amplify a region of the pol gene corresponding to protease and the first half (amino acids 1-240) of reverse transcriptase. Purified amplicons were sequenced using the ABI Prism BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and sequencing primers AK11, AK12, JA204 and JA205 or PRO3 and PRO4 [22]. Sequencing was performed on an ABI Prism 310 Genetic Analyser using the software program ABI Prism 310 Collection and Sequencing Analysis (Applied Biosystems).…”

Antiretroviral Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Protease from Paired Cerebrospinal Fluid and Plasma Samples

“…Two separate regions of the HIV-1 pol gene, one encoding RT amino acids 30-230 and the other encoding the whole protease, were amplified by reverse transcription-nested polymerase chain reaction (PCR), and crude PCR products were then directly sequenced with infrared-labeled primers, as described elsewhere [14]. Reconstruction experiments with mixtures of titrated cloned templates showed that mixed populations are unequivocally detected, provided that they represent at least 10%-20% of the total population, depending on the bases involved in the polymorphism and the composition of the neighbor region [14]. Mutations involved in specific drug resistance were retrieved from the HIV-1 resistance database [15].…”

Longitudinal study in HIV/HCV-coinfected HAART-naive patients and role of HCV genotype

“…139-118). Termination products were electrophoresed and analyzed in a Model 4000L Licor DNA sequencer (Zazzi et al, 1998).…”